Targeted drug or diagnostic compound delivery offers many beneficial effects, including increased drug efficacy, lesser side effects, and better diagnostic capabilities. Numerous compounds have been developed to target specific marker molecules, but additional or more versatile and effective targeting compounds are always needed.
Numerous studies have shown that the formation of new vasculature (angiogenesis) is required for progression of malignant tumors and secondary lesion formation as a result of metastasis. Angiogenesis depends on vascular endothelial cell proliferation, migration, and invasion. A family of adhesion molecule receptors known as integrin receptors regulates these processes. Among the various members of the integrin family, αvβ3 plays a very significant role in angiogenesis. Integrin αvβ3 is minimally expressed on resting or normal blood vessels, but its expression is significantly up-regulated on vascular cells within human tumors. Angiogenesis can be inhibited by anti-αvβ3 antibody. Additionally, antagonists of αvβ3 may significantly inhibit angiogenesis induced by cytokines and solid tumor fragments. Clinical trials of a monoclonal antibody against αvβ3 (Vitaxin™, MedImmune, Inc., Gaithersburg, Md.) showed that the treatment appeared safe and potentially active. However, heterogeneous responses were observed even within the same patient (i.e. some metastases remained stable while others progressed).
For clinical trials relating to αvβ3, a surrogate marker or noninvasive imaging technique that could assess different receptor levels and different activities among different metastases would be extremely helpful in evaluating early treatment responses and understanding the mechanisms behind different responses. It has been demonstrated that MRI imaging of solid tumor angiogenesis may be accomplished using a liposomal paramagnetic imaging agent targeted to endothelial αvβ3 via the anti-αvβ3 monoclonal antibody LM609.
Others have used radiolabeled RGD-containing peptide, which binds to αvβ3, to image tumor cells expressing αvβ3. For example, 125I-labeled 3-iodo-tyr-cyclo(Arg-Gly-Asp-tyr-Val) exhibits high affinity and selectivity for the αvβ3 integrin. Additionally, 18F-labeled RGD-containing glycopeptide has been used for noninvasive PET imaging of αvβ3 expression. Specifically, a cyclic pentapeptide, cyclo(Lys-Arg-Gly-Asp-phe) (“c(KRGDf; SEQ ID NO. 3)” or “cyclic RGD peptide”) was used. The D-Phe construction in this molecule produces a characteristic βII′-turn at the RGD site in a kinked conformation responsible for the αvβ3 integrin binding selectivity. Furthermore, the Lys group may be derivatized at the β-NH2 position without changing the spatial structure of the peptide and thus the binding affinity to the αvβ3 integrin. Further PET studies using 18F-labeled RGD peptides have demonstrated high and specific uptake of the radiotracer in human tumor xenograft.
Matrix metalloproteinases (MMPs) are a family of enzymes capable of degrading the constituents of the extracellular matrix and the basement membrane. There is growing evidence that the MMPs are important not only in tumor invasion and metastases, but also in the creation and maintenance of a microenvironment that facilitates growth and angiogenesis of tumors at primary and metastatic sites. MMPs are up-regulated in virtually all human and animal tumors as well as in most tumor cell lines. Recent studies indicate that expression of MMPs is in general more common in nearby stromal cells than in tumor cells. Many MMPs are induced in connective tissue cells, including fibroblasts and inflammatory cells, as a response to a tumor. These results suggest that MMPs are important contributors to tumor progression and provide the rationale for developing new cancer drugs and diagnostic agents that target MMP activity.
There are reports that suggest that expression of MMPs may have diagnostic or prognostic value. For example, a small study has suggested that serum gelatinase A (an MMP) levels were higher in men with prostate cancer than in men with benign prostatic hypertrophy or normal prostates. In colon cancer samples, immunohistochemical detection of interstitial collagenase (another MMP) is associated with a poor prognosis independent of Duke's stage. These observations are significant because they not only link MMPs with aggressive malignant progression, but they also suggest that tumor-related expression of MMPs may provide important prognostic information that could help direct therapeutic recommendations, including the possibility of targeting inhibition of one or more MMPs.
Additionally, the ability to non-invasively image MMP expression, if properly developed, may be helpful in monitoring treatment responses to MMP inhibitors. Many small molecules containing both hydroxamate and non-hydroxamate zinc binding sites, as well as natural products such as tetracyclines and their derivatives, were developed as MMP inhibitors. Several MMP inhibitors have been tested for clinical trials in cancer patients. However, marimastat and batimastat, two promising MMP inhibitors, have been disappointing in a Phase III clinical trial. Clinical studies with MMP inhibitors have lacked endpoint assessment of MMP-inhibitory activity and the relationship between target modulation and clinical response. This, coupled with disappointing results obtained with MMP inhibitors, suggests that standard clinical trial endpoints are insufficient for the evaluation of molecularly targeted cytostatic agents. Accordingly, there is a great need for the ability to analyze drug efficacy through surrogate markers for MMP expression and/or MMP activity using non-invasive imaging techniques.
It has been suggested that the effect of MMP inhibitors may be stage-specific and tumor-specific. Recent studies support a role for MMPs in earlier stages of the tumor progression continuum. For example, batimastat treatment decreases the number of intestinal adenomas and pancreatic islet cell tumors in the min and RIP-Tag mouse models, respectively; however, tumor burden is diminished in RIP-Tag mice only if the drug is administered before the emergence of large invasive carcinomas. When batimastat is given at advanced tumor stages, no efficacy is observed. These studies establish a spatial and temporal significance for MMPs during tumorigenesis. An analysis of the expression patterns of MMPs in the cancer type and stage and its correlation with treatment outcome may allow a more rational decision on the selection of specific MMP inhibitors and optimal treatment schedule. Additionally, given the importance of MMPs early in tumor development, imaging compounds focused on MMPs are needed.
Using phage display technology, Pasqualini and colleagues have identified a cyclic decapeptide, c(CTTHWGFTLC; SEQ ID NO. 1) (FIG. 1), which not only exhibits selective and potent inhibition of MMP-2 (gelatinase A) and MMP-9 (gelatinase B), but also homes to tumor vasculature in vivo. However, this peptide is very unstable. It degrades completely in cell culture medium in three hours (FIG. 2), preventing its practical use in vivo. This instability is likely due to the use of a disulfide bond to cyclize the polypeptide. More stable peptides are needed for MMP targeting.